USP 62 Nonsterile Product Test for Specified Microorganisms

It is not enough to know how many microbes are in a test sample.  It is important to know what is in the product, what type of microbe.  There are countless objectionable organisms, but USP breaks the variety into groups of basic indicator species:

  • Pseudomonas aeruginosa
  • A Gram negative aerobic rod, P. aeruginosa is an opportunistic pathogen, typically infecting the pulmonary system, urinary tract, burns and wounds.  It is a ubiquitous microbe, found in soil, water, and on skin. 

    P. aeruginosa is an indicator organism for other gram negative non fermenting rods, and other species found that do not exactly match this ID should also be considered as objectionable, depending on the drug.  One such related objectionable microbe is Burkhoderia cepacia, a biofilm forming antimicrobial resistant strain, and subject of many recent recalls.

    10 gm of product is required to perform the test.  P. aeruginosa can be tested with the same 10 gm of product which was used for S. aureus, if both organisms are tested concurrently.

  • Escherichia coli
  • E. coli is well known as a pathogen, it is often in the news causing outbreaks of  food poisoning.  It is a Gram negative, facultative anaerobic coliform, commonly found in the intestine of mammals and birds.  Most strains are harmless, but E. coli is also one of the most diverse species of bacteria, and those strains that produce shiga toxin are particularly lethal.  The important point regarding E. coli is that it is a coliform, or fecal contaminant, and clearly indicates that the drug product that was not made under GMP conditions.

    Any other coliform whose identify is confirmed while conducting the USP test for E. coli may be considered objectionable.  These include Enteroobacter sp., Klebisella sp., Citrobacter sp., and others.

    10 gm of product is required to perform the test.  E. coli can be tested with the same 10 gm of product which was used for Salmonella, if both organisms are tested concurrently.

  • Staphylococcus aureus
  • Any microbial enumeration test must be validated to show that any organisms in the product can actually be cultured.  It is always possible that antimicrobial properties of the product will inhibit the growth of a microbe to a visible colony forming unit.  Validation or “suitability” is accomplished by inoculating plated product with low levels (<100 cfu) of test species and demonstrating equivalence to recovery of that organism in the absence of the product.  Microbial enumeration methods are considered suitable if product results are 50 – 200% of control results.

  • Salmonella
  • Salmonella are Gram negative, facultatively anaerobic rods.  Like E. coli, these are gut bacteria of the Family Enterobacteriaceae.  There are over 2500 serotypes of Salmonella.  Typically invade only the GI tract and causes food poisoning, but they can also be invasive and causes typhoid and paratyphoid fever.  Organic products and excipients such as starch are tested for presence of Salmonella.

    10 gm of product is required to perform the test.  Salmonella can be tested with the same 10 gm of product which was used for E. coli, if both organisms are tested concurrently.

  • Candida albicans
  • C. albicans  is a yeast and like the other pathogens can be a common member of human flora.  It is detected in about half of healthy adults.  However, under the right conditions it can cause an overgrowth of the fungus, as is seen in thrush. 

    Again, C. albicans is only an indicator species, and there are other objectionable yeasts that should be considered while performing this test, including C. tropicalis, C. galabrata, and C. parapsilosis.

    10 gm of product is required to perform the test.

  • Clostridia
  • Clostridia are Gram positive spore forming anaerobes and are some of the most toxic microbes on earth.  Botulism and tetanus are both caused by anaerobic species.   The GI tract of neonates are is not acidic enough to kill Clostridia species.  Clostridia spores in honey have been known to grow in the infant GI tracts and slowly produce lethal botulinum toxin.   Natural products given to newborns should be screened for Clostridia.   Clostridium difficile infection is another problem Clostridia causing tens of thousands of deaths in USA each year. 

    10 gm of product is required to perform the test.

Acceptance Criteria:

Route of Administration

Total APC (cfu/g)

Total Y/M (cfu/gm)

Pathogen Absence

Nonaqueous Oral

<2000

<200

E. coli

Aqueous Oral

<2000

<20

E. coli

Rectal

<2000

<200

--

Oromucosal

<200

<20

S. aureus  and aeruginosa

Gingival

<200

<20

S. aureus and P. aeruginosa

Cutaneous

<200

<20

S. aueus and P. aeruginosa

Nasal

<200

<20

S. aureus and P. aeruginosa

Auricular

<200

<20

S. aureus and P. aeruginosa

Vaginal

<200

<20

S. aureus, P. aeruginosa, and C. albicans

Patches

<200

<20

S. aureus and P. aeruginosa

Inhalation

<200

<20

S. aureus, P. aeuginosa, and Bile tolerant Gram Negative Bacteria